Periodontology 2000, Vol. 52, 2010, 38–52
2010 John Wiley & Sons A/S
Printed in Singapore. All rights reserved
Subgingival biofilm formation
M A S A E K U B O N I W A & R I C H A R D J. L A M O N T
The human body contains numerous distinctive
ecosystems that provide a unique environment for
colonizing microorganisms. The periodontal pocket
is one such microniche. This environment is partially
sheltered from the physical shear forces in the oral
cavity and contains the hard, nonshedding surfaces
of the tooth root along with the shedding surfaces of
gingival mucosa. The junctional epithelium, which is
attached to the tooth root, is poorly differentiated,
lacks keratinization and has relatively wide intercel-
lular spaces. Consequently, junctional epithelium is
permeable and allows the migration of polymor-
phonuclear leukocytes into the periodontal pocket.
Furthermore, the tissues in the periodontal pocket
are bathed in gingival crevicular fluid, a serum
exudate with antioxidant properties. The initial
bacterial colonizers attach to the available surfaces,
as discussed elsewhere in this volume of Periodon-
tology 2000. Later colonizers attach to the antecedent
organisms and assemble into polymicrobial com-
munities. The biofilms on the hard surfaces develop
into spatially organized structures that can extend
several hundred micrometers from the surface. By
contrast, the epithelial surfaces, which are continu-
ally being sloughed and replenished, tend to be
colonized with monolayers of microorganisms.
However, several of the more pathogenic species of
bacteria are able to invade the gingival cells and
tissues where they can remain viable and thus
constitute a nidus of infection.
Interspecies adherence interactions help to shape
the temporal and spatial development of the complex
bacterial consortia in the gingival crevice. Bacteria
within these communities encounter high cell
densities and, in consequence, community living
involves adaptation to higher (and unevenly distrib-
uted) levels of metabolic by-products, secondary
metabolities and other secreted molecules, and to the
sporadic availability of nutrients and oxygen. Bacte-
rial inhabitants of biofilms are known to both col-
laborate (e.g. through nutritional cross-feeding) and
38
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PERIODONTOLOGY 2000
compete (e.g. through production of bacteriocins) as
they strive to optimize their adaptation to these
environmental constraints. Bacteria can also com-
municate with one another through a variety of
sensing and response systems based on either cell-to-
cell contact or detection of soluble mediators. The
signaling molecules are processed through transcrip-
tional and post-transcriptional networks and they al-
low bacterial inhabitants of biofilms to coordinate
activities at a group or community level. An under-
standing of the mechanisms of subgingival biofilm
formation and development needs, therefore, to
accommodate the multiple interspecies interactions
that occur in polymicrobial communities.
Co-adhesion controls community
architecture
The predominant early colonizers of the subgingival
plaque biofilms are the Actinomyces species and
streptococci (110). A complex microbial community
then develops within the space of only a few days
(76), and the secondary colonizers tend to be the
more pathogenic species such as Porphyromonas
gingivalis, Tannerella forsythia, Treponema denticola,
Fusobacterium nucleatum and Aggregatibacter ac-
tinomycetemcomitans. These later colonizers express
numerous adhesions that enable attachment to the
earlier bacterial inhabitants of the region, often
!choosing" partners that are metabolically compati-
ble. Moreover, a number of the secondary colonizers,
in particular F. nucleatum and P. gingivalis, can bind
both to early colonizers and to other, later, colonizers
(46, 113), thus contributing a bridge or node function
to the developing polymicrobial consortia. A surface
configuration that presents multivalent adhesins,
along with multiple adhesins with distinct specificity,
as found on P. gingivalis, for example, will favor
community development.

The importance of co-aggregation or co-adhesion
for the development of plaque biofilms has been
demonstrated in vivo. Slots & Gibbons (95) reported
that the introduction of P. gingivalis into the mouths
of human volunteers resulted in the organism locat-
ing almost exclusively on preformed, streptococcal-
rich supragingival plaque. A close spatial association
between streptococci and Veillonella, and between
streptococci and Actinomyces – pairs of organisms
that co-aggregate in vitro – has been visualized in
developing plaque communities in vivo (11, 12, 71,
72). The ability of potential periodontal pathogens to
locate and attach to compatible antecedent coloniz-
ers may therefore drive the development of patho-
genic subgingival plaque.
Mechanisms of interspecies
binding
A number of studies addressing co-aggregation
among subgingival organisms have started to reveal
the mechanistic basis of these interactions. F. nucle-
atum binds to P. gingivalis through a galactose-spe-
cific lectin-like adhesin that recognizes the sugar
moiety in the capsule and lipopolysaccharide of
P. gingivalis (44, 45, 84). Galactose-containing
receptors for attachment to F. nucleatum are also
provided by the serotype-specific O polysaccharide of
A. actinomycetemcomitans (85) and by the carbohy-
drate moieties on the major outer sheath protein of
T. denticola (83). Moreover, as an illustration of the
multiplicity of adhesin expression, an arginine-inhi-
bitable adhesin (RadA) of F. nucleatum is responsible
for co-adhesion with oral streptococci and accumu-
lation into mixed-species biofilms (39). Hence,
binding of F. nucleatum to streptococci will not oc-
cupy all of the fusobacterial adhesins, and so this
configuration of adhesins will allow fusobacteria–
streptococci consortia to recruit additional gram-
negative pathogens.
S. gordonii P. gingivalis
(189.4 µm) y
x (189.4 µm)
(21.5 µm) z
Subgingival biofilm formation
T. denticola and P. gingivalis have been shown to
accumulate into dual-species biofilms. Attachment
and accumulation requires functional T. denticola
flagella, while the long (FimA) fimbriae and Arg-gin-
gipain (Rgp) B of P. gingivalis also play important
roles in biofilm formation (112). Leucine-rich repeat
proteins of T. denticola and T. forsythia participate in
interbacterial binding with each other and with
F. nucleatum (34, 93).
P. gingivalis –Streptococcus gordonii
One of the best characterized interspecies co-adhe-
sion systems is the binding of the periodontal
pathogen P. gingivalis to substrata of S. gordonii.
This interaction may occur on supragingival surfaces
and, indeed, P. gingivalis is now known to be a
common inhabitant of the supragingival biofilm (28,
58, 100, 110), and can even be detected supragingi-
vally in the absence of subgingival colonization (111).
Consequently, P. gingivalis will be able to establish a
foothold on the supragingival tooth surface, from
where colonization of the subgingival area can occur
by spreading proliferation or by translocation of
dislodged progeny. Alternatively, or concomitantly,
the interbacterial binding interaction may occur
subgingivally, as S. gordonii and related streptococci
are common and abundant constituents of subgin-
gival plaque (27, 96, 110, 111). Accumulation of
P. gingivalis occurs on the streptococcal substrate in
the absence of significant growth and division (57),
and thus represents a means by which the biomass of
P. gingivalis in a community can increase through
attachment and recruitment of cells from the
planktonic phase (Fig. 1).
P. gingivalis adhesins
Co-adhesion between P. gingivalis and S. gordonii is
mediated by two sets of adhesion–receptor pairs: the
Merged
Fig. 1. Confocal microscopy of hete-
rotypic Porphyromonas gingivalis–
Streptococcus gordonii communities.
S. gordonii stained with hexidium
iodide (red) was cultured on glass
plates. P. gingivalis stained with
fluorescein (green) was reacted with
the S. gordonii biofilms for 24 h. The
colocalized bacteria appear yellow in
the merged image. The upper panel
shows x–y projection and the lower
panel shows x–z projection.
39
Kuboniwa & Lamont
long (major) and short (minor) fimbrial subunit
proteins of P. gingivalis that interact with strepto-
coccal glyceraldehyde-3-phosphate dehydrogenase
and Ssp surface proteins, respectively (49–51, 56, 75)
(Fig. 2). The long fimbriae are composed of the FimA
structural subunit protein and extend approximately
3 lm from the cell surface. fimA is part of a gene
cluster that includes the downstream genes fimC,
fimD and fimE, which encode minor components of
mature fimbriae (70). FimE is required for the
assembly of FimC and FimD onto the fimbrillin
(FimA) fiber (70). The two genes upstream of fimA
are involved in the regulation of fimA expression
under the control of the FimS–FimR two-component
system (32, 69). Expression of fimA is also controlled
by the levels of FimA protein itself and by the Rgp
and Kgp gingipains (106). The expression of fimA
responds to environmental cues relevant to condi-
tions in the subgingival area, such as temperature
and hemin concentration (2, 105). The FimA–glycer-
aldehyde-3-phosphate dehydrogenase interaction is
the initial contact event that allows localization of
P. gingivalis on the streptococcal surface (50). The
binding domains of FimA that mediate attachment to
streptococci are localized to a C-terminal region
spanning amino acid residues 266–337 (1).
The short fimbriae of P. gingivalis are approxi-
mately 6.5 nm wide and 103 nm long, and are com-
posed of the Mfa structural subunit protein (75).
Similarly to fimA, mfa is also part of a gene cluster;
however, the roles of the downstream gene products
in the biogenesis of the short fimbriae remain to be
determined. The Mfa protein engages the Ssp pro-
teins on the streptococcal cell surface and increases
the avidity of binding to be more resistant to shear
forces. Mfa–Ssp interactions also initiate a signal
transduction cascade within P. gingivalis that pre-
pares the cells for community living (described later).
Moreover, as the P. gingivalis–S. gordonii commu-
nity develops, the expression of mfa is down-regu-
lated, presumably reflecting differing adhesin
requirements of the organism as the streptococcal
substrate becomes unavailable to P. gingivalis arriv-
ing later (74).
While FimA and Mfa facilitate the accumulation of
P. gingivalis on streptococcal substrates, other
P. gingivalis surface molecules can act to constrain
community development. For example, InlJ, a
member of the cysteine-rich leucine-rich repeat in-
ternalin proteins (86), retards the development of
P. gingivalis–S. gordonii communities (9). The avail-
ability of surface effectors that can either promote or
reduce community development may allow P. gingi-
40
A
P. gingivalis
S. gordonii
FimA Signal transduction
Mfa
Saliva-coated surface
GAPDH
SspA/B
B
P. gingivalis
S. cristatus
FimA
Signal transduction
Mfa
Bacterial-coated surface
ArcA
Fig. 2. Schematic (not to scale) representation of differing
community-relevant events that occur following the
binding of Porphyromonas gingivalis to Streptococcus
gordonii or to Streptococcus cristatus. (A) S. gordonii cells
attach to the saliva-coated tooth surface. S. gordonii pro-
duces multiple adhesins, many of which have cognate
salivary receptors; for simplicity only SspA ⁄ B is shown.
Initial localization of P. gingivalis with S. gordonii is
mediated by the interaction of FimA with glyceraldehyde-
3-phosphate dehydrogenase on the streptococcal surface.
Higher-affinity binding occurs after engagement of Mfa
with SspA ⁄ B. This interaction initiates a signal transduc-
tion event that modulates the P. gingivalis transcriptome.
The resulting phenotypic adaptation of P. gingivalis, along
with the production of signaling molecules, allows the
recruitment of additional P. gingivalis cells from the
planktonic phase and the initiation of community devel-
opment. (B) S. cristatus is a later colonizer of tooth surfaces
and attaches to other organisms. Contact with arginine
deiminase on the surface of S. cristatus induces the down-
regulation of fimA in P. gingivalis and the long fimbriae are
lost. Consequently, community formation does not occur
between P. gingivalis and S. cristatus.
valis to fine-tune the extent of accumulation
according to environmental conditions (as discussed
in greater detail below).

The streptococcal contribution to
community development
The Ssp adhesins (SspA and SspB) are major surface
proteins of S. gordonii and members of the Ag I ⁄ II
family that is widely distributed in the oral
streptococci (37). The SspA and SspB polypeptides
are encoded by tandemly arranged, monocistronic
chromosomal genes and are independently
expressed (19). The Ssp proteins also mediate
attachment of S. gordonii to the salivary pellicle and
their expression is up-regulated by saliva (22),
increasing the receptor availability for P. gingivalis.
The SspA and SspB proteins are structurally con-
served and comprise seven discrete regions: a signal
peptide; an N-terminal region; alanine-rich repeat
blocks; a divergent or variable central region; proline-
rich repeat blocks; a C-terminal region; and a cell-
wall anchorage domain (37). Structure–function
analyses on the mechanism of the Mfa–SspB inter-
action identified a discrete region of SspB, designated
the SspB adherence region, which spans amino acid
residues 1167–1193. The SspB adherence region is
fully conserved between SspA and SspB and is nec-
essary for attachment to P. gingivalis cells or purified
Mfa (8). Within the SspB adherence region, residues
N1182 and V1185 of an NITVK motif are essential for
the recognition of SspB by Mfa (20), and these resi-
dues, along with T1184, are not conserved in SpaP,
the Streptococcus mutans homolog of Ssp that does
not bind to Mfa. The NITVK domain is fully con-
served in Streptococcus oralis and Streptococcus san-
guinis, species that also accumulate in dual-species
communities with P. gingivalis. Substitution of basic
amino acids or serine for N1182, and substitution of
hydrophobic residues Ile, Trp or Phe for V1185, en-
hances the degree of P. gingivalis attachment to the
SspB adherence region, suggesting that both elec-
trostatic and hydrophobic interactions contribute to
SspB adherence region–Mfa binding (16). Further-
more, substitution of the a-helix breaking residues
Pro or Gly is detrimental for P. gingivalis adherence,
consistent with the prediction that secondary struc-
ture plays a role in P. gingivalis adherence (16). The
SspB adherence region also possesses a domain
immediately upstream of the NITVK motif that
resembles the eukaryotic nuclear receptor box (17).
Interactions of nuclear receptors with their co-acti-
vating proteins is driven by the association of a
hydrophobic a-helix of consensus sequence LXXLL,
the nuclear receptor box, with a hydrophobic pocket
in the nuclear receptor protein. This initial interac-
tion is stabilized by electrostatic interactions that
Subgingival biofilm formation
form with charged amino acids that flank LXXLL (90).
The SspB adherence region equivalent contains a
predicted hydrophobic a-helix of sequence VXXLL
that is flanked on each side by positively charged
lysine residues. The introduction of amino acids with
the potential to disrupt the secondary structure of
VXXLL reduces the binding activity of the SspB
adherence region, suggesting that the putative a-
helical character of VXXLL is important for the
interaction of the SspB adherence region with Mfa
(17). Furthermore, replacing the lysines that flank
VQDLL with acidic amino acids also reduces activity,
suggesting that the association of VQDLL with Mfa
may be stabilized by a charge clamp.
In addition to adhesins, a number of streptococcal
processes contribute to community development
with P. gingivalis (47). These can be grouped into
broad categories, as follows: (i) intercellular or
intracellular signaling (chorismate-binding enzyme,
pyruvate oxidase, MarR family transcriptional regu-
lator); (ii) cell wall integrity and maintenance of
adhesive proteins [methionine sulfoxide reductase,
UDP-N-acetylmuramoylalanyl-D-glutamate-2,6-dia-
minopimelate ligase (MurE)]; (iii) extracellular
capsule biosynthesis (cell wall polysaccharide bio-
synthesis protein); and (iv) physiology (glutamate
dehydrogenase, ABC transporter ATP-binding pro-
tein, V-type ATP synthase). Deletion of genes
encoding these proteins diminishes heterotypic
community formation (47). Moreover, several of the
genes encoding these proteins are clustered in a
40-kb region on the S. gordonii chromosome. This
cluster also contains bfrA ⁄ B, a two-component sys-
tem, and bglB, a beta glucoside, both of which are
involved in monospecies S. gordonii biofilm forma-
tion (114). As an organism that is adapted to life in
oral communities, S. gordonii may benefit from
homotypic and heterotypic biofilm-related genes
being in relatively close proximity.
Monospecies P. gingivalis
accumulations
While monospecies biofilms are unlikely to be pre-
valent in the subgingival area, the rapid accumula-
tion of P. gingivalis on substrates of other bacteria
will result in localized areas of dense P. gingivalis
cells. Hence, molecules of P. gingivalis that are
found to be important for monospecies biofilm
formation in vitro can be predicted to play a role in
the developing accretions of P. gingivalis in vivo. A
variety of in vitro assays have been utilized to
model the formation of P. gingivalis monospecies
41
Kuboniwa & Lamont
biofilms, ranging from short-term growth in a mi-
crotiter well plate, to more complex longer-term
chemostat studies. Each of these assays shed light
on different aspects of P. gingivalis monospecies
accumulation, but beyond very simple inferences
current understanding does not allow us to con-
textualize the functional roles of the identified
molecules in the temporal development of P. gin-
givalis biofilms.
A number of studies have shown that P. gingivalis
autoaggregation, and by extension the initiation of a
biofilm, is attributable to FimA (48, 102), and that
loss of short fimbriae enhances autoaggregation
(102). Other work suggests that the Mfa fimbriae are
required for autoaggregation and microcolony for-
mation on solid surfaces (54), and hence the role of
the different fimbrial types may be assay- and con-
text-dependent.
In the microtiter plate assay InlJ is required to
initiate monospecies biofilms (10). Contrast this to
the situation for dual-species biofilms (discussed
earlier) where InlJ is detrimental to P. gingivalis
accumulation and it becomes evident that the pro-
cess of biofilm maturation is finely tuned and
nuanced in order to respond rapidly to changing
environmental conditions such as the presence or
absence of different species of bacteria. The uni-
versal stress protein, UspA, is also required for
P. gingivalis biofilm development, both in microtiter
plate assays and in flow cells (13). Conversely, loss
of several gene products results in enhanced biofilm
growth of P. gingivalis. Inhibitors of homotypic
biofilm accumulation include ClpXP, along with
ClpC, and GalE (UDP-galactose 4-epimerase) (10,
64). Components of the Clp stress-response system
will affect the stability or levels of a number of
proteins in P. gingivalis that could impact biofilm
formation. GalE catalyzes the interconversion of
UDP-glucose to UDP-galactose and in its absence
the amount of galactose in lipopolysaccharide, exo-
polysaccharide and on outer membrane proteins,
such as OMP85, will be reduced, which may stim-
ulate biofilm development (64, 65). Loss of a
glucosyltransferase gene has also been shown to
increase monospecies P. gingivalis biofilm in
microtiter plates (18).
Differential regulation in bacterial
communities
Bacteria adapt to community living through orches-
trated patterns of gene regulation. Global expression
42
analyses using proteome or transcriptome ap-
proaches can provide insights into these complex
systems and begin to reveal the distinct characteris-
tics of community-adapted cells.
Proteome and transcriptome of
monospecies P. gingivalis communities
A proteomic approach has been used to compare
envelope proteins of planktonic P. gingivalis cells
with those of cells cultured as a community in a
chemostat (3). Twenty-four proteins increased in
abundance and 18 decreased significantly in the
biofilm state. Interestingly, the levels of many pro-
teins that were classified into the cell-surface-located
C-terminal domain family increased in the biofilm
cells. These included RgpA, HagA, InlJ, thioredoxin,
CPG70 carboxypeptidase, API extracellular protease
and the Pg99 immunoreactive protein. The C-termi-
nal domain region is thought to participate in
secretion across the outer membrane and attachment
to the surface of the cell, probably via glycosylation
(67, 88, 91). As C-terminal domain proteins are sur-
face exposed, they are thus likely to play important
roles in P. gingivalis virulence. Other proteins that
exhibited significant changes in abundance include
hemin transport-related proteins (HmuY and IhtB),
metabolic enzymes (glyceraldehyde-3-phosphate
dehydrogenase and fumarate reductase) and several
proteins with unknown function, along with putative
proteins.
Transcriptional changes in P. gingivalis cells under
the same conditions as above have also been inves-
tigated (55). Approximately 18% (377 genes) of the
P. gingivalis genome was differentially expressed in
monospecies community cells relative to planktonic
cells. Of these genes, 191 were up-regulated and 186
were down-regulated. Genes that were down-regu-
lated in biofilm cells included those involved in cell
envelope biogenesis, DNA replication, energy pro-
duction, biosynthesis of cofactors, prosthetic groups
and carriers, fatty acid and phospholipid metabolism,
and central intermediary metabolism. These obser-
vations suggest a decrease in cell replication and
growth rate in biofilm cells. By contrast, a number of
genes encoding transport and binding proteins were
up-regulated in P. gingivalis biofilm cells, as were
several genes predicted to encode proteins involved
in signal transduction and transcriptional regulation.
Correlation between messenger RNA levels (55) and
protein levels (3) was modest, a common observation
in other systems (26) and reflective of the multilevel
control systems that regulate bacterial physiology.

Gene regulation in mixed
P. gingivalis–S. gordonii
communities
As discussed above, P. gingivalis develops biofilm
microcolonies on the substrata of S. gordonii but not
on S. mutans (50). In a transcriptome analysis, 33
genes showed up-regulation or down-regulation with
S. gordonii, and the functions of the regulated genes
were predominantly related to metabolism and en-
ergy production (94). Studies of individual P. gingi-
valis dual-species community-associated genes are
still emerging; however, one gene that has been
investigated in some detail is ltp1 (57). The ltp1
gene encodes a cytoplasmic eukaryotic-type low-
molecular-weight tyrosine phosphatase. Interest-
ingly, although expression of Ltp1 was increased in
P. gingivalis–S. gordonii communities, deletion of the
ltp1 gene, or loss of tyrosine phosphatase activity,
increases the level of P. gingivalis accumulation with
S. gordonii. Hence, the role of Ltp1 phosphatase
activity is to constrain community development, a
process that may serve to minimize exposure to
oxygen or facilitate influx of nutrients and efflux of
waste (77). One mechanism by which Ltp1 func-
tions to control community development is through
down-regulating exopolysaccharide production. Ltp1
activity impacts transcription across several exo-
polysaccharide production loci, including those
involved in K-antigen and anionic polysaccharide
production (73). While exopolysaccharide provides a
protective matrix for bacterial cells (104), it is ener-
getically costly and some organisms terminate poly-
mer secretion at a high cell density (62). In addition,
exopolysaccharide can physically propel individual
cells into a more oxygenated environment (104),
hence there is a possible benefit to the anaerobic
P. gingivalis of exopolysaccharide control mecha-
nisms when in a community structure. Ltp1 also
contributes to the regulation of LuxS-dependent
signaling, a topic discussed elsewhere in this volume
of Periodontology 2000.
Signaling mechanisms within
bacterial communities
Within densely packed subgingival communities
there is ample opportunity for communication
among bacteria that are in close proximity. Such
signaling can be based on direct contact, metabolic
co-operation or on diffusible short-range mediators.
Subgingival biofilm formation
A major class of short-range mediators, the auto-
inducers, will not be discussed here, as they are the
topic of another article in this volume of Periodon-
tology 2000.
Metabolic communication
Subgingival bacteria often have complex nutritional
requirements that can be met, in part, through the
release of a metabolite by another organism in the
community. In addition, closely associated organ-
isms can compile a communal suite of enzymes for
degradation of complex substrates into constituents
that can be metabolized by individual members of
the community. These interactions can be con-
sidered signaling, in the broad sense, in that they
represent sensing and responses to environmental
conditions by the organisms, although the extent to
which cellular responses of participating organisms
extend beyond physiological adaptation to nutrient
availability remains to be determined in many cases.
One well-documented example of such metabolic
communication occurs between P. gingivalis and
T. denticola. In culture together these organisms
combine synergistically to produce more biomass
than the additive amounts in monoculture (25). This
nutritional cross-feeding involves the utilization by
P. gingivalis of succinate produced by T. denticola,
and, in turn, the growth of T. denticola is stimulated
by isobutyric acid generated as a metabolic end
product by P. gingivalis (25). Growth of T. denticola
can also be enhanced by proteinaceous substrates
produced by P. gingivalis (68).
Metabolic support for P. gingivalis is also provided
by F. nucleatum, an organism that can tolerate higher
levels of oxygen than P. gingivalis. When cultured
together under aerated conditions, F. nucleatum can
create a reduced microenvironment that is optimal
for P. gingivalis growth (7, 21). F. nucleatum can also
generate ammonia from glutamic and aspartic acids
– amino acids found in crevicular fluid – thus ele-
vating the pH to levels preferred by P. gingivalis (99).
Metabolic pathways relevant to a !periodontal
disease-causing" phenotype
The in vivo relevance of metabolic communication
networks is supported by animal virulence testing. A
polymicrobial consortium of P. gingivalis, T. denti-
cola, T. forsythia and F. nucleatum induces elevated
alveolar bone resorption in rats compared with
monoinfections (43).
Recently, multivariate machine-learning tech-
niques were utilized for comparing automatically
43
Kuboniwa & Lamont
derived metabolic reconstructions of 266 sequenced
genomes, including those of P. gingivalis, T. denti-
cola and F. nucleatum (41). A link was found between
the potential of microorganisms to cause periodontal
disease and their ability to degrade histidine via three
biological pathways: histidine2 (degradation of histi-
dine to L-glutamate); fnc1 (glutamate fermentation);
and c2 (biosynthesis of 5-formimino-tetrahydrofo-
late). In addition, this association held through a
further comparison with the genomes of T. forsythia
and Prevotella intermedia. These three pathways are
interconnected and result in the complete degrada-
tion of L-histidine to acetate and three moles of
ammonia. Interestingly, two enzymes in the 5-for-
mimino-tetrahydrofolate biosynthesis pathway, FolD
(methylenetetrahydrofolate dehydrogenase) and Fhs
(formate-tetrahydrofolate ligase), in P. gingivalis
were significantly up-regulated when in a community
with S. gordonii (94). Furthermore, we have found
(unpublished information; M. Kuboniwa, M. Hackett
Fig. 3. Potential contribution of Streptococcus gordonii to
the conversion of Porphyromonas gingivalis to a more
virulent phenotype within a community. The chorismate-
binding enzyme (Cbe) of S. gordonii can produce 4-
aminobenzoate (pABA) from chorismate. pABA, which is
acquired by P. gingivalis, can be converted into 5,6,7,8-
tetrahydrofolate (THF). THF can be used to produce
44
and R.J. Lamont) that Fhs is significantly up-regu-
lated by P. gingivalis in a community with S. gordonii
and F. nucleatum, indicating that the community
lifestyle may lead to a more virulent P. gingivalis
phenotype. The basis of community-derived
behavioral changes may lie in metabolic communi-
cation related to the formimino- tetrahydrofolate
biosynthesis pathway (Fig. 3). S. gordonii possesses
Cbe, a chorismate-binding enzyme involved in the
production of 4-aminobenzoate (pABA), a precursor
of tetrahydrofolate (33, 98, 103). P. gingivalis is capa-
ble of utilizing exogenous pABA (115), and so pABA
generated by S. gordonii may facilitate degradation of
histidine and push P. gingivalis towards a more viru-
lent phenotype. Support for this concept is provided
by the finding (discussed above) that the loss of Cbe in
S. gordonii reduces community development with
P. gingivalis (47), as the streptococcal contribution to
the dual-species consortia may no longer be sufficient
for the metabolic needs of P. gingivalis.
5-formimino–THF, which is used in the degradation of
histidine that is associated with increased virulence of
P. gingivalis. Gene numbers are shown for S. gordonii
(SGO) and P. gingivalis W83 (PG) or 33277 (PGN). Genes
transcriptionally upregulated in P. gingivalis in the con-
text of a heterotypic community with S. gordonii are
indicated with red arrows.

Arginine deiminase
While many species of subgingival bacteria engage in
synergistic relationships, a number of examples of
antagonism have also been documented. Antagonis-
tic interactions can be based on the production of
antimicrobial compounds such as bacteriocins or
hydrogen peroxide (see below); however, propagation
of a signal by one species, that is designed specifically
to inhibit colonization of a second species, also
occurs. Streptococcus cristatus is distinct from other
oral streptococci in that it possesses characteristic
tufts of fibrils. Also unlike other oral streptococci,
S. cristatus cells tend to be later colonizers of plaque
and more frequent colonizers of periodontal pockets
where they bind to F. nucleatum and form distinctive
!corn-cob" structures that are readily visible in mature
plaque biofilms (30, 52). Contact between S. cristatus
and P. gingivalis, however, initiates a signal trans-
duction cascade in P. gingivalis that causes down-
regulation of fimA expression and consequently fewer
long fimbriae are present on the cell surface (107)
(Fig. 2). With the reduction in fimbrial adhesin
activity, P. gingivalis is unable to bind to or form
communities on substrata of S. cristatus. Signaling is
mediated by arginine deiminase (ArcA) on the surface
of S. cristatus (109). While ArcA is an enzyme
involved in the arginine metabolism pathway that
converts arginine to ornithine, ammonia and CO2,
the signaling function of ArcA does not depend on
enzymatic activity (109). Although S. gordonii also
expresses ArcA, the ability of S. cristatus to repress
FimA production is related to the elevated expression
of arcA as a result of differences in the cis catabolite
response elements of arcA, and in the expression of
trans-acting regulatory proteins (53). The regulatory
network within P. gingivalis that responds to ArcA
signaling involves both transcriptional and post-
transcriptional control of FimA expression (108).
Regions of the subgingival biofilm that are rich in
S. cristatus may be resistant to colonization of
P. gingivalis.
Hydrogen peroxide
Oral streptococci produce hydrogen peroxide, which,
as a strong oxidant, is toxic to bacteria; however,
streptococci are protected from oxidative self-dam-
age in mixed communities with Actinomyces naes-
lundii (36). Hydrogen peroxide can also act as a
signaling molecule for A. actinomycetemcomitans.
When in coculture with streptococci, A. actinomyce-
temcomitans displays enhanced resistance to killing
Subgingival biofilm formation
by human serum. Hydrogen peroxide is sensed by the
oxidative stress response regulator, OxyR, which then
induces up-regulation of the complement resistance
protein, ApiA, in A. actinomycetemcomitans (79).
Contact-dependent signaling
Contact-dependent signaling between P. gingivalis
and S. gordonii is discussed above. Gene regulation
follows a temporal progression because extended
contact between these organisms results in down-
regulation of the gene encoding the short fimbrial
adhesin Mfa (74). Presumably, once initial adhesion
between P. gingivalis and S. gordonii has been
established, Mfa is no longer required for the accu-
mulation of the community. Similarly, in T. forsythia,
expression of the BspA leucine-rich repeat protein
adhesin is down-regulated following contact with
F. nucleatum or P. gingivalis (35).
Genetic exchange within
communities
Horizontal gene transfer by transformation, conju-
gation or transduction is a principal driver of bacte-
rial evolution. The closely packed environment in
biofilm communities facilitates genetic exchange
among constituent cells (61, 97). The opportunistic
pathogen Pseudomonas aeruginosa, for example, can
undergo extensive genetic diversification during
short-term growth in biofilm communities (4). Fur-
thermore, conjugative plasmids themselves express
factors that induce their planktonic bacterial hosts to
form or enter biofilm communities, which then fa-
vors the transfer of the plasmid (24). The diversity
and adaptability produced by horizontal gene trans-
fer provide a form of biological insurance (4) that can
help biofilm communities to survive in harsh envi-
ronments. Subgingival biofilms have been less
extensively studied; however, there are several
mechanisms by which horizontal gene transfer may
be operational.
Mobile genetic elements
Mobile genetic elements can be exchanged
promiscuously between a broad spectrum of
bacteria and contribute to bacterial genome
plasticity. Mobile genetic elements include insertion
sequences, transposons, integrons, bacteriophages,
genomic islands, plasmids and combinations of
these elements.
45
Kuboniwa & Lamont
Conjugative transposons are genetic elements
capable of excision from the chromosome of the
donor genome, transfer to a recipient cell by conju-
gation and insertion into the resulting transconju-
gants" genome (82). Some conjugative transposons
are widespread in oral bacteria. Tn916 and its deriv-
atives, for example, have been found in, or have been
introduced into, more than 50 different species of
bacteria, including the streptococci, Veillonella
parvula, A. actinomycetemcomitans and F. nuclea-
tum (14, 59, 80, 81, 89).
The integron–gene cassette system is a mechanism
that allows bacteria to accumulate diverse genes at a
common locus. Integrons associated with plasmids
or transposons have contributed to the increase in
antibiotic resistance in many gram-negative patho-
gens as a result of their ability to acquire, rearrange
and spread antibiotic-resistance genes. The basic
machinery of an integron is a site-specific recom-
binase of the IntI family, its cognate recombination
site and promoters for the expression of intI and
captured genes. Collectively, these give an integron
the potential to both accumulate gene cassettes and
express the cassette-encoded genes (29). Interest-
ingly, the T. denticola ATCC 35405 genome sequence
contains a 65 kb region containing a number of
open-reading frames hypothesized to have been
acquired by lateral transfer (92), and an unusual
integron (InTde35405) covering 58 kb of this region
has been identified (15).
Genomic islands are regions of the genome ac-
quired horizontally. Base composition analysis (G+C
content, genome signature, codon usage) can be used
to identify laterally transferred genes (40); Table 1
shows genomic islands that have been identified in
periodontally relevant microbes using base compo-
sition analysis and BLAST taxonomy data [Oralgen
database (http://www.oralgen.lanl.gov/)]. Subsequ-
ent BLASTP homology analysis with Bacteroides
CTn341 and CTnDOT revealed that three periodontal
pathogens (T. forsythia ATCC43037, P. intermedia 17
and P. gingivalis W83) have predicted genomic
islands that correspond to the tra gene cluster, which
is the DNA transfer region in CTn341 and CTnDOT
(5, 60).
DNA-transfer mechanisms in
P. gingivalis
Recently, Naito et al. (63) presented the whole gen-
ome sequence of P. gingivalis ATCC 33277, a strain
better adapted for oral colonization and induction of
bone loss than strain W83 (42, 78). Comparison
46
between W83 and ATCC 33277 revealed 461 ATCC
33277-specific and 415 W83-specific predicted pro-
tein coding sequences. In addition, 175 regions with
genomic re-arrangements were observed between
the two strains. Both strains contained large numbers
of mobile elements, such as conjugative transposons,
insertion sequences and miniature inverted-repeat
transposable elements. In ATCC 33277, there are four
copies of conjugative transposons, designated as
CTnPg1-a, CTnPg1-b, CTnPg2 and CTnPg3, all of
which are different from conjugative transposon-
related gene clusters in W83. CTnPg1-a contains 50
coding sequences, including a set of genes for
conjugative transfer and integration, and several of
these show moderate sequence homologies to the
genes of CTn341 and CTnDOT. The other conjugative
transposons (CTnPg1-b, CTnPg2 and CTnPg3) were
truncated and disrupted by multiple insertion
sequences.
Besides conjugative transposons, a total of 93
insertion sequence elements and 48 miniature in-
verted-repeat transposable elements were found in
ATCC 33277. Insertion sequences are the simplest
transposable elements and can be as short as 600–
700 bp, simply encoding a transposase. The presence
of several closely related insertion sequence elements
in the genome allows homologous recombination
between unrelated elements, provided that each of
the elements carries a copy of the same insertion
sequence element. The insertion sequence elements
identified in ATCC 33277 were classified into six
types, ISPg1–ISPg6 , all of which are also present in
W83 (66). Miniature inverted-repeat transposable
elements comprise a group of small mobile genetic
elements. They do not encode transposases by
themselves but have terminal inverted repeats that
are the same as, or very similar to, those of some
insertion sequence elements, and they are thus
transposable by the action of transposase provided
in trans by the cognate insertion sequence element.
Functional DNA transfer in P. gingivalis was stud-
ied by Tribble et al. (101). P. gingivalis strains ATCC
33277, 381, ATCC 49417, A1A7-28 and a low-passage
clinical isolate (MP4-504), were able to transfer the
Bacteroides–Escherichia coli shuttle vectors, pT-COW
and pFD340, to E. coli by a mechanism most con-
sistent with conjugation. By contrast, strains W83,
W50 and another clinical strain, 5083, did not transfer
either plasmid at detectable levels. Horizontal trans-
fer of genomic DNA between P. gingivalis W83 and
ATCC 33277 was also demonstrated and, moreover,
in contrast to plasmid DNA conjugation, both strains
were able to transfer chromosomal DNA to each

Table 1. Genomic islands in periodontal microbes*
Organism
Porphyromonas gingivalis W83
Tannerella forsythia ATCC43037
Prevotella intermedia 17
Aggregatibacter actinomycetemcomitans
HK1651
Treponema denticola ATCC 35405
Fusobacterium nucleatum ATCC 25586
*Compiled from the Oralgen database (http://www.oralgen.lanl.gov/).
CRISPR, clustered regularly interspaced short palinormic repeats; IS, insertion sequence; LOS, lipooligosaccharide.
Subgingival biofilm formation
Number of distinct genomic islands Description
1 Bacteroides conjugative transposon-
related island (tra gene cluster)
1 Hemagglutinin-related cluster
1 Thiamin biosynthesis cluster
1 Potassium uptake gene cluster
1 Transport-related genomic island
1 Mobilization cluster, ISPg-related
2 Mobilization cluster
3 Uncharacterized genomic island
5 IS-related genomic island
12 IS-related potential island
1 Bacteroides conjugative transposon-
related island (tra gene cluster)
1 Conjugative transposon-related genomic
island
1 Transport-related genomic island
1 Hemolysin-related genomic island
1 Thermolysin-related genomic island
1 Glycosyltransferase-related genomic
island
1 Phage-related genomic island
1 CRISPR-associated genomic island
1 Type I restriction system genomic island
1 Electron transport-related genomic
island
2 Uncharacterized genomic island
1 Bacteroides conjugative transposon-
related island (tra gene cluster)
1 Glycosyltransferase gene cluster
1 N-acetylmuramoyl-L-alanine amidase-
containing cluster
1 ATP synthase and glycosyltransferase
gene clusters
1 Membrane protein gene and ABC trans-
port gene cluster
1 Mobilization gene cluster with fic-related
gene
1 Uncharacterized genomic island with
integrases
3 Uncharacterized genomic island
1 O-antigen biosynthesis and transport
gene cluster
1 Leukotoxin gene cluster
1 Cytolethal distending toxin gene cluster
1 Tight adherence gene cluster
1 LOS biosynthesis enzyme
3 Uncharacterized genomic island
1 Super integron
1 ABC transport system
1 Capsular polysaccharide biosynthesis
cluster
1 sapI-related and hypothetical protein-
containing island
1 Uncharacterized genomic island
0
47
Kuboniwa & Lamont
other. Chimeras showed phenotypic changes in the
ability to accrete into biofilms, implying that DNA-
transfer events are sufficient to generate measurable
changes in complex behaviors.
Transformation and transduction
The conserved ability to acquire DNA molecules by
natural transformation enables access to DNA as a
source of nutrients or to increase genetic variability.
Transformation has not been extensively investigated
in subgingival biofilms; however, some strains of
A. actinomycetemcomitans are naturally competent
(23).
Horizontal gene transfer through transduction
mediated by bacteriophage is responsible for the
lysogenic conversion of many different nonpatho-
genic bacteria, including E. coli, Vibrio cholerae, Lis-
teria spp. and Streptococcus spp., to pathogens (6).
Periodontal bacteria, such as A. actinomycetemcomi-
tans, fusobacteria and T. denticola have been shown
to possess bacteriophage (38, 87, 92). In A. actino-
mycetemcomitans, phage Aa phi 23 correlates with
population genetic structure, but does not appear to
influence virulence (31). The full extent and role of
bacteriophage and transduction in the subgingival
microbiota remains to be determined.
Conclusions
The subgingival biofilm is more than a random
assemblage of organisms seeking shelter from the
hostile environment of the oral cavity. Rather, there
exists sophisticated social networking, based initially
on very specific recognition of surface characteristics,
which provides the discrimination necessary for the
formation of metabolically compatible, physiologi-
cally integrated communities. Community develop-
ment is controlled by programmed patterns of gene
expression and multilevel regulation of protein
expression and activity. Organisms within these
communities continually monitor the host environ-
ment and the nature and intentions of other
organisms that may seek to participate in community
affairs. Interspecies communication systems may al-
low rudimentary group decisions to occur. The sub-
gingival ecosystem is thus a dynamic environment
and it is likely that much community-specific
physiology is devoted to adaptation to stimulate an
increase in biomass or to limit and stabilize accu-
mulation according to prevailing conditions. Once a
degree of stability or maturity is reached, organisms
48
can begin the process of genetic exchange and the
production of genetically diverse daughter cells,
some of which will exhibit increased fitness. The
success of these strategies is evidenced by the fact
that in the absence of host intervention, the subgin-
gival area is colonized by biofilm communities from
shortly after birth until death.
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